Compositions and methods for combination therapy with dengue virus and dendritic cells

ABSTRACT

Described herein are compositions and methods for treating cancer through the combination of tumor antigen-pulsed dendritic cells and Dengue Virus. The combination of the two forms of therapeutic intervention provides enhanced tumor cell reduction compared to either alone. The cancer targeted by compositions and methods described herein may be a solid cancer or blood cancer.

CROSS-REFERENCE

This application is a Continuation of U.S. patent application Ser. No.16/389,445, filed Apr. 19, 2019 which is a Continuation of U.S. patentapplication Ser. No. 16/172,487, filed Oct. 26, 2018, now U.S. Pat. No.10,357,553, which is a Continuation of U.S. patent application Ser. No.15/799,793, filed Oct. 31, 2017, now U.S. Pat. No. 10,159,727, which isa Continuation of U.S. patent application Ser. No. 15/639,632, filedJun. 30, 2017, now U.S. Pat. No. 9,849,167, which is a Continuation ofU.S. patent application Ser. No. 15/200,751, filed Jul. 1, 2016, nowU.S. Pat. No. 9,730,989, which claims the benefit of U.S. ProvisionalApplication No. 62/231,351, filed Jul. 2, 2015, which are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2016, isnamed 48253-702 304 SL.txt and is 1,871 bytes in size.

BRIEF SUMMARY

Provided herein are methods for treating cancer in a subject in needthereof, comprising: incubating dendritic cells (DCs) with at least onetumor cell antigen; administering a Dengue Virus Type 2 serotype strainDENV-2 #1710 to the subject; and administering the DCs to the subject.Further provided herein are methods, wherein the at least one tumor cellantigen is from a solid cancer cell or a blood cancer cell from thesubject. Further provided herein are methods, wherein the blood canceris leukemia or lymphoma. Further provided herein are methods, whereinthe solid cancer is melanoma, breast, prostate, or brain cancer. Furtherprovided herein are methods, wherein the DCs are collected byleukapheresis. Further provided herein are methods, wherein the DCs orthe at least one tumor cell antigen are from the subject. Furtherprovided herein are methods, wherein the DCs and the at least one tumorcell antigen are from the subject. Further provided herein are methods,wherein the at least one tumor cell antigen is from the subject. Furtherprovided herein are methods, wherein the DCs and the at least one tumorcell antigen are from the subject. Further provided herein are methods,wherein the at least one tumor cell antigen is present in a suspensioncomprising single cells. Further provided herein are methods, whereinthe DCs are pulsed with the at least one tumor cell antigen. Furtherprovided herein are methods, wherein the at least one tumor cellsantigen is a peptide. Further provided herein are methods, wherein thepeptide is about 9 amino acids in length. Further provided herein aremethods, wherein the Dengue Virus Type 2 serotype strain DENV-2 #1710 ispresent in an amount sufficient to induce a systemic infection. Furtherprovided herein are methods, wherein the DCs are administered after thesubject's temperature reaches 38 degrees Celsius or higher. Furtherprovided herein are methods, wherein the DCs are administered after thesubject's temperature reaches 38.5 degrees Celsius. Further providedherein are methods, wherein monocytes of the subject are processedobtain the DCs. Further provided herein are methods, wherein the DCs aremature DCs capable of antigen uptake.

Provided herein are methods for treating cancer in a subject in needthereof, comprising: lysing tumor tissue from a subject to form a lysisproduct comprising apoptotic or necrotic bodies; incubating dendriticcells (DCs) with the lysis product; administering a Dengue Virus to asubject; and administering the DCs to the subject after the subject'stemperature reaches 38 degrees Celsius or higher. Further providedherein are methods, wherein the tumor tissue comprises a cell from asolid cancer or a blood cancer. Further provided herein are methods,wherein the blood cancer is leukemia or lymphoma. Further providedherein are methods, wherein the solid cancer is melanoma, breast,prostate, or brain cancer. Further provided herein are methods, whereinthe DCs are from the subject. Further provided herein are methods,wherein the DCs are collected by leukapheresis. Further provided hereinare methods, wherein the DCs are not from the subject. Further providedherein are methods, wherein the tumor tissue is present in a suspensioncomprising single cells prior to lysis. Further provided herein aremethods, wherein the DCs are pulsed with the lysis product. Furtherprovided herein are methods, wherein the Dengue Virus is administered inan amount sufficient to induce a systemic infection. Further providedherein are methods, wherein obtaining DCs comprises processing thesubject's monocytes to produce the dendritic cells. Further providedherein are methods, wherein the DCs are mature DCs capable of antigenuptake. Further provided herein are methods, wherein the tumor tissue isin an amount of 0.5 to 5 grams. Further provided herein are methods,wherein the tumor tissue is in an amount of 1 to 2 grams. Furtherprovided herein are methods, wherein the Dengue Virus is serotype 1, 2,3, 4, or 5. Further provided herein are methods, wherein the DengueVirus is serotype 2. Further provided herein are methods, wherein theDengue Virus is Dengue Virus Type 2 serotype strain DENV-2 #1710.Further provided herein are methods, wherein the Dengue Virus is presentin an amount sufficient to induce a systemic infection. Further providedherein are methods, wherein the DCs are mature DCs capable of antigenuptake. Further provided herein are methods, wherein the DCs areadministered to the subject after the subject's temperature reaches 38.5degrees Celsius.

Provided herein are methods for targeting cancer cells, comprising:incubating dendritic cells (DCs) with at least one tumor cell antigen;administering a Dengue Virus Type 2 serotype strain DENV-2 #1710 to asubject; and administering the DCs to the subject.

Provided herein are methods for targeting cancer cells, comprising:lysing tumor tissue from a subject to form a lysis product comprisingapoptotic or necrotic bodies; incubating dendritic cells (DCs) with thelysis product; administering a Dengue Virus to the subject; andadministering the DCs to the subject after the subject's temperaturereaches 38.5 degrees Celsius or higher.

Provided herein are compositions for reduction of tumor cells, thecomposition comprising an effective amount of Dengue Virus Type 2serotype strain DENV-2 #1710 for initiating an immune response, whereinthe Dengue Virus Type 2 serotype strain DENV-2 #1710 provides for anenhanced reduction of tumor cells when administered prior toadministration of tumor antigen-primed dendritic cells compared toadministration of either agent alone. Further provided herein arecompositions, wherein the tumor cells are from a solid cancer or a bloodcancer. Further provided herein are compositions, wherein the bloodcancer is leukemia or lymphoma. Further provided herein arecompositions, wherein the solid cancer is melanoma, breast, prostate, orbrain cancer.

Provided herein is use of an effective amount of Dengue Virus Type 2serotype strain DENV-2 #1710 for the manufacture of a medicament for thetreatment of cancer. Provided herein is use of an effective amount ofDengue Virus Type 2 serotype strain DENV-2 #1710 for treating cancer.Provided herein is An effective amount of Dengue Virus Type 2 serotypestrain DENV-2 #1710 for use in treating of cancer.

BACKGROUND

Immunotherapy, unlike cytotoxic drugs, radiation, and surgery,stimulates the immune system to recognize and kill tumor cells. Numerousattempts have been made in stimulating the immune system to recognizeand destroy tumor cells. These have been met with limited success due tothe self-identity of peptides selected as target for immunotherapy, lackof immune activation, adverse events, and/or tumor immune evasionmechanisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 exemplifies a method of treatment with Dengue virus and primeddendritic cells.

FIG. 2 shows a plot of corresponding to the number of lung metastasesfrom melanoma cells in mice under various treatment conditions. Thepatterned bars represent the mean number of lung metastases for eachcondition.

FIG. 3 shows a plot of corresponding to the number of lung metastasesfrom melanoma cells in mice under various treatment conditions. Thepatterned bars represent the mean number of lung metastases for eachcondition.

DETAILED DESCRIPTION OF INVENTION

Definitions

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

The term “subject” as used herein includes to mammals. Mammals includerats, mice, non-human primates, and primates, including humans.

Combination Delivery

Described herein are compositions and methods wherein dendritic cellvaccination is combined with an adjuvant effect of a strain of Denguevirus (DV) to overcome tumor immune evasion mechanisms and deplete tumorcells. Methods described here may be used to treat a subject 101 forcancer by obtaining 101 dendritic cells 102 and tumor cells 104 from thesubject, exposing the dendritic cells to the tumor cells or tumor celllysate 105, also referred to as “pulsing” the dendritic cells, to primed(or “activated”) the dendritic cells, delivering 107 the resultingprimed and tumor-targeting dendritic cells to the subject after thesubject has had his/her immune system stimulated with DV 108 (see, e.g.,FIG. 1). Optionally, the tumor antigen is not from the subject can beused for pulsing the dendritic cells.

Tumor immune evasion mechanisms are responsible for the lack of efficacyseen with most immunotherapy platforms. Compositions and methodsdescribed herein provide for a multi-pronged approach, combiningphysiological (hyperthermic reduction of tumor perfusion), immunological(activation of effector cells of the adaptive and innate immune system),and apoptosis-inducing pathways (sTRAIL) to destroy tumor cells. UsingDV as an adjuvant to activate many pathways working in synergy maysupport the eradication of mutated tumor cells, improving the clinicalefficacy of the cancer immunotherapy. Methods described herein providecancer immunotherapies based on multiple mechanisms of action in concertand result in a decline in the ability of the tumor cells to employresistance methods compared to delivery of any single method along.

Disclosed herein are methods for treating cancer in a subject in needthereof, comprising: obtaining dendritic cells (DCs); incubating the DCswith at least one tumor cell antigen; administering a Dengue Virus Type2 serotype strain to the subject; and administering the DCs to thesubject. In some instances, the Dengue Virus Type 2 serotype strain isDENV-2 #1710. In some instances, the dendritic cells are autologousdendritic cells. In some instances, the dendritic cells are allogeneicdendritic cells. In some instances, incubating the DCs with at least onetumor antigen comprises incubating the DCs with a tumor cell. In someinstances, incubating the DCs with at least one tumor antigen comprisesincubating the DCs with a tumor cell lysate.

Methods of Isolating and Lysing Tumor Cells

Provided herein are methods for combination therapy with a DV andactivated DCs to target tumor cells. In some instances, DCs are primedwith tumor cells from a subject. In some instances, the tumor cells areisolated cells from a tumor microenvironment of the subject, referred toherein as tumor supporting cells. In some instances, dendritic cells areexposed to/pulsed with tumor cells, tumor supporting cells and/orpeptides thereof, such that the dendritic cells will target tumor cellsand/or tumor supporting cells that support tumor growth and metastasis(e.g. endothelial cells, vascular cells, immune cells, etc.). In someinstances, peptides/antigens from tumor cells and tumor supporting cellsinduce dendritic cells or cytotoxic lymphocytes with receptors forpeptides/antigens on both tumor cells and tumor supporting cells,resulting in targeting of the dendritic cells or cytotoxic lymphocytesto the tumor microenvironment rather than only the tumor cells. In someinstances, tumor cells and/or tumor supporting cells are obtained from abiopsy of tumor tissue. In some instances, the biopsy comprises cellsselected from tumor cells, adipocytes, fibroblasts, endothelial cells,infiltrating immune cells, and combinations thereof.

Provided herein are methods for combination therapy with a DV andactivated DCs to target tumor cells, wherein the DCs are activated withlysed tumor cells and/or tumor supporting cells and surroundingextracellular matrix. In some instances, lysing comprises contacting thetumor cells and/or tumor supporting cells with an NH₄Cl enzyme solutionto eliminate red blood cells. In some instances, the lysing comprisescontacting the tumor cells and/or tumor supporting cells withhypochlorous acid solution to induce immunogenic cell death. In someinstances, the cells are lysed gently enough to not destroy peptides. Insome instances, the cells are lysed to produce apoptotic or necroticbodies. In some instances, the methods comprise lysing the tumor cellsand/or tumor supporting cells with an enzymatic solution. In someinstances, the methods comprise lysing the tumor cells and/or tumorsupporting cells with a peroxide-free solution or a lowperoxide-containing solution. In some instances, the methods compriselysing the tumor cells and/or tumor supporting cells with a detergentsolution. In some instances, the detergent is selected from, but is notlimited to, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween20, Tween 80, octyl glucoside, octyl thioglucoside, SDS, CHAPS, andCHAPSO. In some instances, the detergent solution is purified ofperoxides, and other impurities. In some instances, the detergent isabout 0.1% to about 10% v/v of the detergent solution. In someinstances, the detergent is about 0.1% to about 5% v/v of the detergentsolution. In some instances, the detergent is about 0.5% to about 5% v/vof the detergent solution. In some instances, the detergent is about 1%to about 10% v/v of the detergent solution. In some instances, thedetergent is about 1% to about 5% v/v of the detergent solution. In someinstances, the methods comprise lysing cells without shaking, vortexing,freezing, thawing, shear pressure, sonicating and/or heating the cells.

Methods of Isolating and Priming Dendritic Cells (DCs)

Provided herein are methods for combination therapy with a DV andactivated DCs to target tumor cells, wherein the DCs comprise allogeneicor autologous dendritic cells. In some instances, methods describedherein comprise administering allogeneic primed dendritic cells to asubject. In some instances, methods described herein compriseadministering autologous primed dendritic cells to a subject.

In some instances, methods described herein comprise obtaining dendriticcells from CD34⁺ progenitor cells in the bone marrow. In some instances,methods described herein comprise obtaining dendritic cells fromCD1⁺CD14⁺ immature monocytes in the peripheral blood. In some instances,obtaining the dendritic cells comprises leukapheresis. In someinstances, leukapheresis comprises withdrawing a unit of blood from thesubject or a donor, separating a series of blood-components: red cells,platelets, and most of the plasma factors, which are returned to thesubject, with the white blood cells remaining. In some instances,methods described herein comprise testing the white blood cells forsterility, shipping or storing them cold (4° C.), and or processing theDC from the apheresis product.

In some instances, methods described herein comprise pulsing DCs with apeptide. In some instances, the methods comprise pulsing DCs with apeptide that binds MHC Class I molecules (“MHC Class I peptide”). Insome instances, methods described herein comprise pulsing DCs with apeptide that binds MHC Class II molecules (“MHC Class II peptides”). Insome instances, methods described herein comprise pulsing DCs with MHCClass I peptides and MHC Class II peptides. In some instances, thepulsing makes the DCs competent to prime CTL and target CTL to tumors.In some instances, methods described here comprise pulsing DCs withmanufactured/synthetic Class I and/or Class II peptides. In someinstances, the Class I and/or class II peptides are manufactured, thenadded to the DC medium, optionally in in microgram quantities or less.In some instances, methods described herein include Class II peptidesfor a sustained immune response. In some instances, methods describedherein comprise DNA or RNA sequencing of the peptide (i.e. tumorantigen) and/or using electroporation to insert the DNA or RNA into theDC to trigger antigen processing. In some instances, methods describedherein do not require HLA matching of DCs. In some instances, thepeptide or portion thereof is represented by an amino acid sequenceselected from EGSRNQDWL (SEQ ID NO:1), (TAYRYHLL) (SEQ ID NO: 2), orcombinations thereof.

In some instances, methods described herein comprise contacting thedendritic cells with autologous tumor cells or lysates thereof. In someinstances, methods described herein comprise contacting the dendriticcells with autologous whole-tumor cells (e.g. tumor cells and tumorsupporting cells) or lysates thereof which contain the full array ofpotential antigens for a broad and deep immune response. In someinstances, methods described herein comprise contacting the dendriticcells with tumor cell lysate comprising apoptotic or necrotic bodies. Infurther instances, the tumor cell lysate comprises tumor antigens fromthe microenvironment surrounding the tumor cells, such as extracellularmatrix proteins. In some instances, contacting comprises pulsing,wherein pulsing comprises contacting the dendritic cells more than onceat one or more intervals. In some instances, “pulsing” of DCs comprisescontacted DCs with peptides/antigens, tumor cells, tumor supportingcells, tumor cell lysate and/or tumor supporting cell lysate. In someinstances, lysing the tumor cells does not comprise trypsin enzymedigestion and freeze-thaw cycles, which are simple and fast, but candamage the delicate peptides within. In some instances, methodsdescribed herein comprise using an automated cell processor, like theMiltenyi GentleMACS system, by way of non-limiting example, to manuallymince the sample; suspend cells in PBS solution; and/or separate thetumor cells with a pre-selected tissue-specific software-controlledrotor system. In some instances, the single-cell suspension ismembrane-lysed with minimal damage to tumor peptides. In some instances,methods described herein comprise pulsing the DCs from about 1 hour toabout 24 hours. In some instances, methods described herein comprisepulsing the DCs from about 12 hours to about 48 hours. In someinstances, methods described herein comprise pulsing the DCs from about8 hours to about 24 hours. In some instances, methods described hereincomprise pulsing the DCs for about 18 hours.

Dengue Viruses

Provided herein are methods for combination therapy with a Dengue virus(DV) and activated DCs to target tumor cells, wherein the DV isadministered to a subject. As used herein, the term “Dengue virus”includes any serotype of Dengue virus serotypes 1, 2, 3, 4, or 5.

Dengue viruses are Arboviruses, and are transmitted exclusively bymosquitoes of the Aedes aegypti and albopictus species. The virus has acomplex life cycle involving an unidentified forest-dwelling mammalianreservoir (possibly primates), and human hosts. The female mosquitotakes a blood meal from an infected person, the virus replicates to ahigh infectious titer (105/ml), in gut epithelial cells, then istransmitted to another person when the mosquito withdraws its styletusing back pressure after another blood meal. Dengue epidemics infect 50million persons annually, with several thousand deaths, usually childrenwith inadequate treatment of secondary infection-related shock.

The Dengue virus genome encodes structural proteins, capsid protein C,membrane protein M, envelope protein E, and nonstructural proteins, NS1,NS2a, NS2b, NS3, NS4a, NS4b and NS5. In some instances, the Dengue virusis a live strain of the Dengue virus. In some instances, the Denguevirus is an attenuated strain of the Dengue virus. In some instances,the Dengue virus is a weakened strain of the Dengue virus. In someinstances, the Dengue virus is selected the following serotypes ofdengue virus: DENY-1, DENV-2, DENV-3, DENV-4, and DENV-5, andcombinations thereof.

Dengue Viruses are positive-strand RNA viruses of the Togavirus Family,sub-family Flaviviridae, (Group B). The virus has an icoashederalgeometry and is approximately 40-45 nanometers in diameter. The 11,000base genome codes for a nucleocapsid (NC), protein, a prM membranefusion protein, an envelope glycoprotein I, and 5 non-structuralproteins NS1-NS5. The NC protein forms the viral core, with the envelopespikes attached via the prM complex. The E glycoprotein is the maintarget of neutralizing antibodies, and the NS-3 and NS-4 proteins makeup the main targets for CD4+and CD8+CTL.

The dengue viruses make up five distinct serotypes, DENV-1 throughDENV-5. The serotypes 2 and 4 are cross-neutralizing for IgG, and types1 and 3 are also cross-neutralizing. Immunity is not complete, however,and dengue is unique among viral infections in that a subsequentinfection by a non-cross-neutralizing serotype carries an increased riskof mortality due to shock syndrome from immune hyper-activation.

Provided herein are compositions and method using such compositions,wherein the composition comprises Dengue virus serotype 1, 2, 3, 4, or5. In some instances, the DV is serotype 2. In some instances the DVserotype 2 is DENV-2 strain #1710. The strain is from a sample takenfrom Puerto Rico in 1985 and characterized as type A from a restrictionsite specific RT-PCR analysis using 4 primers (see Table 1) specific tothe envelope gene region. See Harris et al., Virology 253, 86-95 (1999).Restriction site specific RT-PCR with these primers producesamplification products of 582 base pairs, 754 base pairs, and possibly676 base pairs. The DENV-2 strain #1710 is recorded in a CDC database asentry number 555. See Harris (1999). The DENV-2 strain #1710 wasisolated during a Puerto Rican epidemic. This outbreak had 9,540suspected cases of DV, with one suspected, but no confirmed deaths dueto the virus, which indicates the toxicity of DEN-2 strain #1710 is verylow and therefore suitable for the methods disclosed herein.

TABLE 1  Sequence and Position of Primers to Amplify DENV-2 virusesGenome Primer Sequence Position Strand RSS1 5′-GGATCCCAAGAAGGGGCCAT-3′1696-1715 + (SEQ ID NO: 3) RSS2 5′-GGCAGCTCCATAGATTGCT-3  2277-2259 −(SEQ ID NO: 4) RSS3 5′-GGTGTTGCTGCAGATGGAA-3′  1524-1542 +(SEQ ID NO: 5) RSS4 5′-GTGTCACAGACAGTGAGGT-3′  2371-2353 −(SEQ ID NO: 6)

DV has many characteristics supporting its use as a potentimmune-stimulant in cancer immunotherapy. DV has affinity for immatureB-lymphocytes and antigen-presenting cells (APC) of monocyte/macrophageand DC lineage. A unique feature of DV is that primary infections resultin activation of a T_(H)1-type response of CD4+ and CD8+ helper-inducerand cytotoxic-effector CTL. By infecting, but not killing the APC, DVup-regulates their CD80 and CD83 expression, resulting in apro-inflammatory T_(H)1 cytokine profile. Primary DV infections induce aT_(H)1 type response with activated CD4+and CD8+effector T cells as wellas LAK cells. This type of response is seen in patients having completeresponses to cancer immunotherapies (see Table 2).

TABLE 2 Tumor immune evasion mechanisms and DV infection Immune evasionDengue counter-attack Low levels of MHC on tumor cell Hi Interferon-γraises MHC levels by up-regulating MHC gene expression prevent CTLrecognition Point mutations in Tumor Peptides LAK/CIK cells target“escaped” tumor cells expressing aberrant peptides or MHC prevent TCRbinding Tumor vessels lack factors for CTL Hi [TNF-α] restores gaps byaltering PECAM-1, restores ICAM-1/VCAM-1 attachment and traffickingexpression and P and E-selectins FasL can kill Fas⁺ CTL by Hi [IL-6, 15]protects Fas⁺ CTL by up-regulating FLIP ligand triggering apoptosisHLA-G protects from NK Cells Hi [IL-2, 7, 12, 15] raise activation of NKStromal barriers inhibit CTL Hi [IFN-γ] activates Macrophages to M₁Myeloid-Derived Suppressor Cells, iNKT Cells can decrease MDSC (MDSC)CTL inactivated by TGF-β T_(H)1 cytokines reactivate tolerant CTL TumorPI-9 blocks CTL killing Hi [CD8] & ICAM-1 expression can restorelow-avidity CTL recognition and lysis by stabilizing weak interactionsbetween TCR and MHC + self-peptide T-regulatory cells block CTL HiCD4^(Helper) cells overcome CD4^(Reg) cells

In primary infections, the death rate from DV is very low (1 in 61,000per Manson's Tropical Diseases). The virus infects but does not kill APCof the monocyte-macrophage and Dendritic Cell lineage. These infectedAPC then begin a cytokine cascade of the pro-inflammatory (TNF-alpha andIL-1 beta), and T_(H)1 (IL-2, IL-7, IL-12, IL-15, and IL-21) types.These cytokines result in strong activation of both the adaptive (CTL)and innate (NK) immune systems. After a 3-5 day incubation period, thefever rises to 39.5-40.5° C., and remains elevated for 4-5 days. Thepatient experiences intense headache, joint pain, malaise, andsensitivity to light. A rash covering the chest back and sometimes legsand arms develops by day 3 of fever. Clinically, dengue infectionsresult in lowered platelet counts leading to hemorrhage, which rangesfrom minor to life-threatening in case of shock syndrome. With propersupportive care based on judicious fluid management, recovery iscomplete in 99% of cases.

Expression of genes in cells of the subject may be increased by DVinfection, including, but not limited to, IL-1 beta, IL-2, IL-7, IL-12,IL-15, IFN-alpha, IFN- gamma, TNF-alpha, TNF-beta, GM-CSF, CD8 antigen,ICOSLG, CCL3, CCLS, TRAIL, IP10, GNLY, GZMA, HLA-DRA, HLA-DP alpha1,HLA-DP beta 1, and ZAP70. Increased levels of proteins corresponding tothese genes may be observed in circulating fluids of the subject. Levelsmay be increased at least 2-fold. Levels may be increased between 2-foldand 1000-fold. Levels may be increased between 2-fold and 100-fold.Levels may be increased between 2-fold and 10-fold. Cell types of asubject administered DV4 may be increased by DV infection, including,but not limited to, CD8+CD44+62L-cells, CD4+CD44+ CD62L^(lo) cells,HLA-DR+ CD8+ cells, Tia-1 CD8+ cells, VLA-4 CD8+ cells, ICAM-1 CD8+cells, and LFA-1 CD8+ cells. In some instances, TNF-α, is released bythe immune system during DV infection. TNF-α is an inflammatory cytokinewith pleiotropic effects, including direct killing of tumor cells viaTRAIL (TNF-Apoptosis-Inducing-Ligand).

In some instances, DV induces high levels of soluble TRAIL (sTRAIL) froma variety of cells including γδCTL, activated M1 macrophages andplasmacytoid DC (pDC). In some instances, DV activates IFNβ, amultifunctional cytokine with a 10-fold higher affinity for the samereceptor as IFNα. IFNβ has similar antiviral properties in suppressingtranscription of viral RNA, but is much more potent than IFNα ininducing apoptosis in tumor cells. Nitric oxide and IFNβ could act in asynergistic fashion during dengue infection. These molecules may work intandem to overcome resistance to apoptosis mediated by the high levelsof sTRAIL induced by M₁ macrophages, pDC, and δγ CTL.

Cancer

Methods described herein provide for treating a subject for cancer.Methods described herein also provide for clearing cancer cells. In someinstances, administering DV to the subject induces an immune response.In some instances, the immune response is potent as compared to a commonvirus, such as a common cold virus. In some instances, the immuneresponse results in tumor regression.

DNA microarray analyses have revealed that hundreds of geneticallydistinct tumor clones may exist in a single patient with advanced tumor.There is a pattern of negative correlation between O₂ supply and geneticmutation rates. The majority of agents such as cytotoxic drugs,antibodies, and small molecules, are nearly always blood-borne, exertinga Darwinian selective pressure to tumor clones that evade therapeuticmechanisms. Clones with the lowest perfusion rates have both low drugexposure and high capacity to evade immune system detection, making themresistant to conventional therapies. Provided herein are methods forcancer cell targeting, comprising inducing fever hyperthermia byadministering DV to the subject with cancer, starving low-flow,resistant clones with mutated phenotypes, leaving more geneticallystable clones for elimination by activated lymphocytes and other arms ofthe immune system. In some instances, the methods comprise combiningfever with activation of CTL and lymphokine-activated killer cells (LAK)by administering pulsed DCs, lead to higher response rates than withconventional cancer therapies (e.g. antibody drug conjugates, kinaseinhibitors, small molecules, etc.) or CTLs alone. The immune suppressionseen in patients with advanced cancer is a complex and dynamic process.It involves tolerance to the tumor antigens themselves, which areusually recognized as “self” by CTL. In some instances, methodsdescribed herein comprise breaking this tolerance and achieving highlevels of T_(H)1 cytokines, which DV infection induces.

Cancers targeted herein may be a recurrent and/or a refractory cancer.In some instances, the cancer is an acute cancer or a chronic cancer. Insome instances, the cancer is an accelerated refractory cancer. In someinstances, the cancer is in remission. In some instances, the cancer isa stage I, stage II, stage III, or stage IV cancer. In some instances,the cancer is a juvenile cancer or adult cancer. Examples of cancersinclude, but are not limited to, sarcomas, carcinomas, lymphomas orleukemias. In some instances, the cancer is a solid tumor or aliposarcoma.

In some instances, the cancer is a sarcoma. The sarcomas may be a cancerof the bone, cartilage, fat, muscle, blood vessels, or other connectiveor supportive tissue. In some instances, sarcomas include, but are notlimited to, bone cancer, fibrosarcoma, chondrosarcoma, Ewing's sarcoma,malignant hemangioendothelioma, malignant schwannoma, bilateralvestibular schwannoma, osteosarcoma, soft tissue sarcomas (e.g. alveolarsoft part sarcoma, angiosarcoma, cystosarcoma phylloides,dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, and synovial sarcoma). The sarcoma may comprise aEwing's sarcoma.

In some instances, the cancer is a carcinoma. Carcinomas are cancersthat begin in the epithelial cells, which are cells that cover thesurface of the body, produce hormones, and make up glands. By way ofnon-limiting example, carcinomas include breast cancer, pancreaticcancer, lung cancer, colon cancer, colorectal cancer, rectal cancer,kidney cancer, bladder cancer, stomach cancer, prostate cancer, livercancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer,uterine cancer, oral cancer, penile cancer, testicular cancer,esophageal cancer, skin cancer, cancer of the fallopian tubes, head andneck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneousor intraocular melanoma, cancer of the anal region, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, cancer ofthe urethra, cancer of the renal pelvis, cancer of the ureter, cancer ofthe endometrium, cancer of the cervix, cancer of the pituitary gland,neoplasms of the central nervous system (CNS), primary CNS lymphoma,brain stem glioma, and spinal axis tumors. In some instances, the canceris a skin cancer, such as a basal cell carcinoma, squamous, melanoma,nonmelanoma, or actinic (solar) keratosis.

In some instance, the cancer is a neuroendocrine cancer. In someinstances, the cancer is a pancreatic cancer, thyroid cancer, or aprostate cancer. In some instances, the cancer is an epithelial cancer,breast cancer, endometrial cancer, ovarian cancer, stromal ovariancancer, or cervical cancer. In some instances, the cancer is a skincancer. In some instances, the cancer is a neo-angiogenic skin cancer.In some instances, the cancer is a melanoma. In some instances, thecancer is a kidney cancer, a lung cancer. Exemplary lung cancersinclude, without limitation, a small cell lung cancer or a non-smallcell lung cancer. In some instances, the cancer is a colorectal cancer,e.g., a gastric cancer or a colon cancer. In some instance, the canceris a brain cancer. In some instances, the cancer is a brain tumor. Insome instances, the cancer is a glioblastoma or an astrocytoma.

In some instances, the cancer is a lung cancer. In some instances, thelung cancer is a non-small cell lung carcinoma (NSCLC), small cell lungcarcinoma, or mesotheliomia. Examples of NSCLC include squamous cellcarcinoma, adenocarcinoma, and large cell carcinoma. In some instances,the mesothelioma is a cancerous tumor of the lining of the lung andchest cavity (pleura) or lining of the abdomen (peritoneum). In someinstances, the mesothelioma is due to asbestos exposure.

In some instances, the cancer is a central nervous system (CNS) tumor.In some instances, the CNS tumor is classified as a glioma or nonglioma.In some instances, the glioma is malignant glioma, high grade glioma,diffuse intrinsic pontine glioma. Examples of gliomas includeastrocytomas, oligodendrogliomas (or mixtures of oligodendroglioma and15mmune1515oma elements), and ependymomas. Astrocytomas include, but arenot limited to, low-grade astrocytomas, anaplastic astrocytomas,glioblastoma multiforme, pilocytic astrocytoma, pleomorphicxanthoastrocytoma, and subependymal giant cell astrocytoma.Oligodendrogliomas include low-grade oligodendrogliomas (oroligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomasinclude meningiomas, pituitary adenomas, primary CNS lymphomas, andmedulloblastomas. In some instances, the cancer is a meningioma.

In some instances, the cancer is a blood cancer. In some instances, thecancer is leukemia. In some instances, the cancer is a myeloid leukemia.In some instances, the cancer is a lymphoma. In some instances, thecancer is a non-Hodgkin's lymphoma. In some instances, the cancer isselected from myelogenous leukemia, lymphoblastic leukemia, myeloidleukemia, an acute myeloid leukemia, myelomonocytic leukemia,neutrophilic leukemia, myelodysplastic syndrome, B-cell lymphoma,burkitt lymphoma, large cell lymphoma, mixed cell lymphoma, follicularlymphoma, mantle cell lymphoma, 15mmunel5 lymphoma, recurrent smalllymphocytic lymphoma, hairy cell leukemia, multiple myeloma, basophilicleukemia, eosinophilic leukemia, megakaryoblastic leukemia, monoblasticleukemia, monocytic leukemia, erythroleukemia, erythroid leukemia andhepatocellular carcinoma. In some instance, the cancer is ahematological malignancy. In some instance, the hematological malignancyis a B cell malignancy. In some instance, the cancer is a chroniclymphocytic leukemia. In some instance, the cancer is an acutelymphoblastic leukemia. In some instance, the cancer is a CD19-positiveBurkitt's lymphoma. In some instance, the leukemia is an acutelymphocytic leukemia, acute myelocytic leukemia, chronic lymphocyticleukemia, or chronic myelocytic leukemia. Additional types of leukemiasinclude, but are not limited to, hairy cell leukemia, chronicmyelomonocytic leukemia, and juvenile myelomonocytic leukemia.

In some instance, the lymphoma develops from a B lymphocyte or Tlymphocyte. Two major types of lymphoma are Hodgkin's lymphoma,previously known as Hodgkin's disease, and non-Hodgkin's lymphoma. Insome instance, the Non-Hodgkin lymphoma is indolent. In some instance,the Non-Hodgkin lymphoma is aggressive. Non-Hodgkin's lymphomas include,but are not limited to, diffuse large B cell lymphoma, follicularlymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small celllymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma,mediastinal large B cell lymphoma, Waldenström macroglobulinemia, nodalmarginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma(SMZL), extranodal marginal zone B cell lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, and lymphomatoidgranulomatosis.

Methods of Administration

Provided herein are methods comprising administering Dengue virus anddendritic cells to a subject in need thereof In some instances, thevirus is provided in an aqueous form. In some instances, the virus islyophilized and reconstituted in an aqueous solution (e.g., salinesolution). In some instances, the virus is administered by a routeselected from subcutaneous injection, intramuscular injection,intradermal injection, percutaneous administration, intravenous (“i.v.”)administration, intranasal administration, intralymphatic injection, andoral administration. In some instances, the subject is infused with thevirus by an intralymphatic microcatheter.

In some instances, the Dengue virus is initially administered at least24 hours before administering the dendritic cells. In some instances,the Dengue virus is initially administered between about 12 hours andabout 96 hours before administering the dendritic cells. In someinstances, the Dengue virus is initially administered between about 24hours and about 72 hours before administering the primed dendriticcells. In some instances, the Dengue virus is initially administeredbetween 1 day and 4 days before administering the primed dendriticcells. In some instances, the Dengue virus is administered only once. Insome instances, the Dengue virus is administered more than once. In someinstances, the Dengue virus is administered only before receivingdendritic cells. In some instances, the Dengue virus is administeredafter receiving the primed dendritic cells. In some instances, theDengue virus is administered before and after receiving the primeddendritic cells.

In some instances, the methods comprise administering the Dengue virusat a dose of about 0.5 ml of 10⁶ pfu/ml. In some instances, the dose isbetween about 10³ pfu/ml and about 10⁸ pfu/ml. In some instances, thedose is between about 10³ pfu/ml and about 10⁶ pfu/ml.

In some instances, successful infection or inoculation of the subjectwith the Dengue virus is confirmed by the development of hyperthermia orfever. In some instances, successful infection or inoculation of thesubject with the Dengue virus is confirmed by the presence or increaseof circulating cytokines in the blood/plasma of the subject. Cytokinesmay include, but are not limited to, interleukin-2 and interferon-gamma.

In some instances, methods described herein comprise administeringprimed dendritic cells to a subject in need thereof only once. In someinstances, the primed dendritic cells are administered more than once.In some instances, the primed dendritic cells are administered a firsttime and a second time, wherein the first time and the second time areseparated by about 1 day, about 2 days, about 3 days, about 4 days,about 5 days, or about 6 days, about 8 days, about 10 days, aboutl2days, or about 18 days. In some instances, the first time and the secondtime are separated by about 1 week, about 2 weeks, about 3 weeks, orabout a month. In some instances, the first time and the second time areseparated by more than a month. In some instances, the first time andthe second time are separated by less than 12 months. In some instances,the first time and the second time are separated by more than 12 months.

In some instances, methods described herein provide for administeringprimed dendritic cells to a subject when the subject is hyperthermic. Insome instances, primed dendritic cells are administered after thesubject has spike a fever. In some instances, primed dendritic cells areadministered after the subject's temperature has risen to between about37.5° C. and about 42° C. In some instances, the primed dendritic cellsare administered after the subject's temperature has risen to betweenabout 38° C. and about 42° C. In some instances, the primed dendriticcells are administered after the subject's temperature has risen to atleast about 38.5° C. In some instances, the primed dendritic cells areadministered after the subject's temperature has risen to 38.5° C. Insome instances, the subject's temperature is measured by a tympanic ororal method.

EXAMPLES Example 1 Generation and Pulsing of Dendritic Cells (DC)

A method described by Lutz M., et. Al. (J.Immunol.Methods 223:77-92,1999), was employed to generate mature DC form mouse bone marrow. Bonemarrow suspensions were incubated in petri dishes in medium supplementedwith recombinant murine GM-CSF for 10 days. Non-adherent cells werecollected, centrifuged and resuspended in medium containing GM-CSF andlipopolysaccharide. Two days later, the DCs were harvested and theirviability was determined by trypan-blue exclusion. Purity of the DC wasdetermined by flow cytometry analysis. DCs were pulsed with thesynthetic peptides at 10 μg/ml for 18 hours. After 18 hours ofincubation, DCs were harvested, washed twice in HB SS, and resuspendedin HESS for additional studies (see Example 2 and 3).

Example 2 Dengue Virus and Dendritic Cells for the Treatment of Melanomain a First Mouse Model

A mouse model assay was performed to observe results from combinationtargeting of cancer cells using a Dengue virus (DV) strain and tumorantigen primed dendritic cells (DCs). DV C57BL/6 mice were inoculatedwith 0.05 ml of Dengue virus (DEN-2 strain #1710) at 1×10⁶ or1×10⁷pfu/ml by injection in the base of tail. Recombinant murine IL-2(Genzyme) and IFN-gamma (Sigma Pharmaceuticals) were administered byintravenous infusion at 2,000 (rIL-2) and 500 1U (rIFN-gamma) on days 5,10, 15, and 20 following administration of Dengue virus (DEN-2 strain#1710, CDC database entry number 555, provided by Dr. Duane Gubler).Seven days after the Dengue virus administration, C57BL/6 mice wereimmunized with mouse DC incubated with the 2 peptides separately andinjected intravenously. Peptides were synthesized. The H-2b-restrictedpeptide from Ovalbumin (OVA-8), SIINFEKL (SEQ ID NO: 7), was used as acontrol. B16 melanoma-associated H-2b-restricted peptides derived fromthe antigens gp100/pme117 (EGSRNQDWL (SEQ ID NO: 1)) and from TRP-1/75(TAYRYHLL (SEQ ID NO: 2)) were used to pulse murine DC (see Example 1for details). Two additional immunizations with DC were given at 14-dayintervals. Three days after the last DC infusion, mice were challengedwith 5×10⁴ viable B16 melanoma cells intravenously in the lateral tailvein and then followed for survival, which was recorded as thepercentage of surviving animals over time (in days) after tumorinjection. Data was recorded from five or more mice/group (see Table 3and FIG. 2).

TABLE 3 MOUSE NO. OF LUNG Condition Group ID METASTASES Mean DV10⁶pfu/ml + 2 × 10⁶ DC pulsed with gp100/TRP2 2 II-2-1 55 DV10⁶ pfu/ml + 2× 10⁶ DC pulsed with gp100/TRP2 2 II-2-2 68 DV10⁶ pfu/ml + 2 × 10⁶ DCpulsed with gp100/TRP2 2 II-2-3 57 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed withgp100/TRP2 2 II-2-4 62 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed with gp100/TRP22 II-2-5 52 58.8 No DV + 2 × 10⁶ DC pulsed with gp100/TRP2 1 II-1-1 58No DV + 2 × 10⁶ DC pulsed with gp100/TRP2 1 II-1-2 62 No DV + 2 × 10⁶ DCpulsed with gp100/TRP2 1 II-1-3 66 No DV + 2 × 10⁶ DC pulsed withgp100/TRP2 1 II-1-4 72 No DV + 2 × 10⁶ DC pulsed with gp100/TRP2 1II-1-5 60 63.6

The number of lung metastases observed in mice administered in Group 2(Dengue Virus serotype 2 strain #1710 and tumor peptide primed DCs) was7.5% lower than control mice in Group 1, administered the tumor peptideprimed DCs without the Dengue virus.

Example 3 Dengue Virus and Dendritic Cells for the Treatment of Melanomain a Second Mouse Model

A mouse model assay was performed to observe results from combinationtargeting of cancer cells using a Dengue virus (DV) strain and tumorantigen primed dendritic cells (DCs). Mice were administered cytokinesto parallel the response to DV observed in humans.

Tumors were established in mice using the H-²b-restricted B16 murinemelanoma cells line (ATCC #CRL-6322). Peptides (B16 melanoma associatedH-²b-restricted peptides derived from antigens gp100/pme117 and fromTRP-1/gp75) used for pulsing the dendritic cells were synthesized.Dendritic cells were generated from mouse bone marrow according to themethod described in Lutz et al. (J. Immunol. Methods 223:77-92, 1999).

On day 0, mice received 5×10⁴ viable B16 melanoma cells intravenously inthe lateral tail vein to establish pulmonary metastases. On day 7, themice were inoculated with 0.05 ml of Dengue virus (DEN-2 strain #1710,CDC database entry number 555) at 1×10⁶ or 1×10⁷pfu/ml by injection inthe base of tail. Recombinant murine IL-2 (Genzyme) and IFN-gamma (SigmaPharmaceuticals) were administered by intravenous infusion at 2,000 1U(rIL-2) and 500 1U (rIFN-gamma) at 5-day intervals followingadministration of Dengue virus (DEN-2 strain #1710). On days 21, 35 and49, the mouse DC were incubated with the 2 peptides separately andinjected intravenously in 2 sequential administrations on the same dayto match the route and schedule of administration in patients (seeExample 2 for additional details). Control groups of mice received noDengue virus or dendritic cells pulsed with H-²b-restricted peptide fromovalbumin (OVA-8), SIINFKEL Treatment and control groups are shown inTable 4.

TABLE 4 Dengue Virus # of dendritic cells and type of peptide Group A10⁶ pfu/ml 10⁶ DC pulsed with gp100/pme117 (EGSRNQDWL) (SEQ ID NO: 1)10⁶ DC pulsed with TRP-1/gp75 (TAYRYHLL) (SEQ ID NO: 2) Total 2 × 10⁶ DCpulsed with peptide/mouse Group B 10⁶ pfu/ml 10⁷ DC pulsed withgp100/pme117 (EGSRNQDWL) (SEQ ID NO: 1) 10⁷ DC pulsed with TRP-1/gp75(TAYRYHLL) (SEQ ID NO: 2) Total 2 × 10⁷ DC pulsed with peptide/mouseGroup C - Control None 10⁶ DC pulsed with gp100/pme117 (EGSRNQDWL) (SEQID NO: 1) 10⁶ DC pulsed with TRP-1/gp75 (TAYRYHLL) (SEQ ID NO: 2) Total2 × 10⁶ DC pulsed with peptide/mouse Group D - Control 10⁶ pfu/ml 10⁶ DCpulsed with OVA (SIINFEKL) (SEQ ID NO: 7) 10⁶ DC pulsed with OVA(SIINFEKL) (SEQ ID NO: 7) Total 2 × 10⁶ DC pulsed with peptide/mouse

On day 90, animals were sacrificed and lung tumor colonies were counted.Pulmonary metastases were enumerated in a blinded, coded fashion afterinsufflation and fixation of the lungs with Fekette's solution. Datawere reported as the mean number of metastases; four mice/group (seeTable 5 and FIG. 3). Histopathology of the following major organ systemswere performed: brain, heart, lungs, liver, kidneys, spleen and gonads(data not shown).

TABLE 5 NO. OF LUNG Condition Group MOUSE ID METASTASES Mean DV10⁶pfu/ml + 2 × 10⁶ DC pulsed with A III-1-1 82 gp100/TRP2 DV10⁶ pfu/ml + 2× 10⁶ DC pulsed with A III-1-2 87 gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DCpulsed with A III-1-3 78 gp100/TRP2 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsedwith A III-1-4 72 gp100/TRP2 79.75 DV10⁷ pfu/ml + 2 × 10⁶ DC pulsed withB III-2-1 87 gp100/TRP2 DV10⁷ pfu/ml + 2 × 10⁶ DC pulsed with B III-2-277 gp100/TRP2 DV10⁷ pfu/ml + 2 × 10⁶ DC pulsed with B III-2-3 92gp100/TRP2 DV10⁷ pfu/ml + 2 × 10⁶ DC pulsed with B III-2-4 85 gp100/TRP285.25 No dengue virus + 2 × 10⁶ DC pulsed with C III-3-1 97 gp 100/TRP2No dengue virus + 2 × 10⁶ DC pulsed with C III-3-2 94 gp 100/TRP2 Nodengue virus + 2 × 10⁶ DC pulsed with C III-3-3 88 gp 100/TRP2 No denguevirus + 2 × 10⁶ DC pulsed with C III-3-4 91 gp 100/TRP2 92.5 DV10⁶pfu/ml + 2 × 10⁶ DC pulsed with OV D III-4-1 180 DV10⁶ pfu/ml + 2 × 10⁶DC pulsed with OV D III-4-2 174 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed with OVD III-4-3 165 DV10⁶ pfu/ml + 2 × 10⁶ DC pulsed with OV D III-4-4 177 174

The number of lung metastases observed in mice in Group C (administeredtumor antigen primed DCs and no virus) was 47% less than control Group D(administered DENV-2 #1710 and DCs exposed to a control peptide). Thenumber of lung metastases observed in mice in Group A (administeredDENV-2 #1710 and tumor antigen primed DCs) was 54% less than controlGroup D (administered DENV-2 #1710 and DCs exposed to a controlpeptide). The number of lung metastases observed in mice in Group B(administered DENV-2 #1710 and tumor antigen primed DCs) was 51% lessthan control Group D (administered DENV-2 #1710 and DCs exposed to acontrol peptide). The average reduction in Group A and B compared toGroup D was 52.8%.

Example 4 Manufacture and Use of Dengue Virus

A Master Cell Bank with validated and certified cell lines from Vero(African Green Monkey Kidney Cells) is set up and tested for absence ofany contaminants and adventitious organisms. Vero lines are used by theWorld Health Organizations to produce a variety of viral vaccines.Dengue virus is passaged in a validated Vero Line derived from theMaster Cell Bank and established as a Working Cell Bank according toguidelines established by the FDA Center for Biologics (CBER). DengueVirus Type 1, 2, 3, or 4 from initial seed stock are added to the VeroCells of the WCB at a MOI of 10⁻⁵.

The first 4-ml overlay medium—containing 1% SeaKem LE agarose (FMCBioProducts, Rockland, Me.) in nutrient medium (0.165% lactalbuminhydrolysate [Difco Laboratories, Detroit, Mich.]), 0.033% yeast extract[Difco], Earle's balanced salt solution, 25 mg of gentamicin sulfate[BioWhittaker, Walkersville, Md.] and 1.0 mg of amphotericin B[Fungizone; E. R. Squibb & Sons, Princeton, N.J.], per liter and 2%FBS)—is added after adsorption of the 200-ml virus inoculum for 1.5 h at37° C. Following incubation at 37° C. for 7 days, a second 2-ml overlaycontaining additional 80 mg of neutral red vital stain (GIBCO-BRL,Gaithersburg, Md.) per ml is added. Plaques are counted 8 to 11 daysafter infection.

A plaque assay on final virus cultures is performed, with a target of10⁶ pfu/ml as a standard dose. Ultracentrifugation is used toconcentrate virus if this target is not reached. Following confirmationof virus titer, final product is filtered to remove any cellular debris,tested for absence of any adventitious organisms, and upon final lotreleased, bottled in 5 ml bottles with a label as a BSL-2 levelpathogen, and stored at 4° C. until ready for shipment andadministration.

Example 5 Manufacture and Use of Dendritic Cells Pulsed with TumorAntigens

Monocytes are separated from other white blood cells (e.g. T cells. Bcells, NK cells, eosinophils and basophils). This is accomplished withimmune-magnetic selection or, alternatively, by adherence properties.Immuno-magnetic selection involves pouring the white blood cells into asterile plastic column with plastic beads coated with antibodies forimmune cell CD surface proteins: (CD4/CD8/CD56, etc.). The unwantedcells adhere to the beads leaving the monocytes to pass through and becollected. In positive selection, magnetic beads coated withCD1+/CD14+capture the monocytes, a magnet is placed against the column,and unwanted cells are flushed out of the column with PBS solution. Themonocytes are then washed off the beads for the next step. In adherenceselection, the properties of monocytes to stick to certain surfaces areused to separate them by running the apheresis product down a slantedcolumn.

Alternatively, bone marrow cells are depleted for lymphocytes and MEWClass positive cells by Fluorescent Activated Cell Sorting (FACS) withmonoclonal antibodies for CD3, CD4, and CD8. Remaining cells arecultured overnight in Iscove's modified Dulbecco's medium (IMDM)supplemented with 10% Fetal Calf Serum (FCS), 2-ME, and 100 IU/mlpenicillin, 1 mM sodium pyruvate, and 10 uM nonessential amino acids at37° C. in a 5% CO2 atmosphere in 24-well plates with approximately 1million cells in 1 ml culture medium/well. After 24 hours, the cells arereplated and cultured in the presence of Granulocyte-Macrophage ColonyStimulation Factor (GM-CSF), and recombinant IL-4 at 900 U/ml. After 3to 4 days, media to be exchanged for fresh cytokine media.

Alternatively, dermal dendritic cells (DDC) are prepared using thefollowing methods: Keratomes from healthy human volunteers are incubatedin a solution of the bacterial proteases Dispase type 2 at a finalconcentration of 1.2 U/ml in RPMI 1640 for 1 hour at 37° C. After theincubation period, epidermis and dermis are easily separated. Epidermaland dermal sheets are then cut into small (1-10 mm) pieces after severalwashing with PBS, and placed in RPMI 1640 supplemented with 10% FetalBovine Serum (FBS), and placed in 10-cm tissue culture plates. After 2-3days, pieces of tissue are removed, and the medium collected. Cellsmigrating out of the tissue sections into the medium are spun down,resuspended in 1-2 ml fresh medium and stained with trypan blue. Furtherenrichment is achieved by separation on a metrizamide gradient. Cellsare layered onto 3-ml columns of hypertonic 14.5% metrizamide andsedimented at 650 g for 10 minutes at room temperature. Low densityinterphase cells are collected and washed in two successively lesshypertonic washes (RPMI 1640 with 10% FBS and 40 mM NaCl) to returncells to isotonicity.

When the monocytes are collected, they may number only a few thousand.The recombinant human growth factors rhuInterleukin-4 (IL-4), andrhuGranulocyte-Macrophage-Colony-Stimulation Factor (GM-CSF), are usedin a multi-step protocol to accomplish the expansion of DC numbers tothe range of 50 million. The addition of IL-4 and GM-CSF expands thenumbers and the development of mature-DC markers: (CD11⁺, CD80⁺, CD83⁺),as well as increased expression of both Class I (for presentation ofshort peptides to CDS⁺, and Class II MEW complexes (for presentation oflonger peptides to CD4⁺Helper-Inducer T lymphocytes). Afterapproximately 4 days, the number of mature DCs will be measured.

Pulsing the Dendritic Cells

Whole autologous tumor cell lysate is prepared by several methods. Thedevelopment of automated cell processors like the Miltenyi GentleMACSsystem allows the sample to be manually minced, suspended in PBSsolution, then a pre-selected tissue-specific software-controlled rotorsystem separates the tumor cells. The single-cell suspension can bemembrane-lysed with minimal damage to tumor peptides. Alternatively, thesample does not separate the tumor cells. Instead the sample is left tocontain tumor cells and supporting cells (e.g. cells from the tumormicroenvironment). Cells are lysed with NH4C1 to eliminate red bloodcells and produce apoptotic and necrotic bodies without destroyingpeptides needed for CTL induction.

After addition of the lysate, the DCs engulf and process the peptidesfor presentation to CTL. The final step is the maturation with aninflammatory signal. A preferred agent is Lipopolysaccharide (LPS) frombacterial cell walls. After exposure to LPS, the DC up-regulate theirCD80/CD83⁺activation markers, increase production of IL-12p70 to inducea Type 1 CTL response, and become resistant to further antigen uptakeand processing.

After final sterility, specificity, and viability testing, the DC aretransferred to polymer bags suitable for freezing at −70° C. in liquidN₂, storage up to 1 year, and shipping to the clinic for use. The bagsare shipped cool overnight, then re-warmed to 37° C. in a warm-waterbath before intravenous administration with a 0.9% NaCL solutionconcurrent. The i.v. DCs traffic to the lungs, where some will betrapped, but the majority will pass to secondary lymphatic organs suchas liver and spleen white pulp T-cell zones to prime the CTL.

Example 6 Combination Delivery for Treatment of Cancer

Administration of the Dengue Virus is similar to that of other viralvaccine injections. A subject has an area of skin in the shoulder(deltoid) region cleaned with alcohol, then 0.5 ml of the virus isinjected under the skin to mimic a mosquito bite. Once the subject has afever the reaches 38.5° C., after 2-3 days from DV injection, thepatient is infused by intralymphatic microcatheter with pulsed (primed)dendritics. Injections are repeated until the patient is negative fordisease. DC fusions will use cells manufactured in Example 5.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for reducing or treating cancer, the method comprising administering to a subject having cancer: an effective amount of Dengue Virus; and dendritic cells, to the subject, wherein the dendritic cells are administered more than once.
 2. The method of claim 1, wherein the dendritic cells are tumor antigen primed.
 3. The method of claim 2, wherein a tumor cell lysate that comprises a tumor antigen is used to prime the dendritic cells.
 4. The method of claim 2, wherein a tumor cell lysate that comprises a non-tumor antigen is used to prime the dendritic cells.
 5. The method of claim 4, wherein the non-tumor antigen is from a cell of the tumor microenvironment.
 6. The method of claim 1, further comprising a second administration of the dendritic cells.
 7. The method of claim 4, wherein the administration and the second administration are separated by at least 3 weeks.
 8. The method of claim 1, wherein the Dengue Virus is of serotype DENV-1, DENV-2, DENV-3, DENV-4, or DENV-5.
 9. The method of claim 8, wherein the Dengue Virus is of serotype DENV-1.
 10. The method of claim 1, wherein the cancer is a solid cancer.
 11. The method of claim 10, wherein the solid cancer is skin cancer.
 12. The method of claim 1, wherein the dendritic cells are autologous.
 13. A method for reducing or treating metastatic cancer, the method comprising administering to a subject having metastatic cancer: an effective amount of Dengue Virus; and dendritic cells, to the subject, wherein the dendritic cells are administered more than once.
 14. The method of claim 13, wherein the reduction or treatment of the metastatic cancer comprises a reduction in metastases.
 15. The method of claim 14, wherein the metastases are in the lungs of the subject.
 16. The method of claim 13, wherein the metastatic cancer is skin cancer.
 17. The method of claim 16, wherein the skin cancer is melanoma.
 18. The method of claim 13, wherein the dendritic cells are autologous.
 19. The method of claim 13, further comprising a second administration of the dendritic cells at least 3 weeks after the administering.
 20. The method of claim 13, wherein the Dengue Virus is of serotype DENY-1, DENV-2, DENV-3, DENV-4, or DENV-5. 